SERCA activity in pancreatic B-cells is usually closely coupled to glucose metabolism and ATP production. Thus, ATP depletion induces SERCA-dependent Ca²⁺-release. We investigated whether ablation of K_ATP channels in SUR1-KO mice influences intracellular Ca²⁺-storage. In wildtype B-cells (WT) treated with 15 mM glucose (15G) 0.5 mM FCCP induced a fast increase in [Ca²⁺]_c from 355±39 to 606±74 nM (n=6) that was completely absent after store depletion with CPA (n=7). By contrast, CPA only diminished Ca²⁺-release in SUR1-KO B-cells (n=13) pointing to recruitment of additional Ca²⁺-stores. Analogous results were obtained in WT B-cells when K_ATP channels were blocked with 1 mM tolbutamide or 50 mM nateglinide. FCCP provoked an increase in [Ca²⁺]_c by 159±6 % (n=5) and 138±13 % (n=5), respectively, despite of SERCA-inhibition. These changes did not depend on high [Ca²⁺]_c or Ca²⁺-influx as they did not occur when continuous Ca²⁺-influx was forced by 10 mM arginine (n=4) or 30 mM K⁺ (n=4) but were induced by additional application of tolbutamide (n=4). In summary, our data demonstrate that genetic or pharmacological ablation of K_ATP channels drastically alters intracellular Ca²⁺-homeostasis by activation of additional Ca²⁺-stores. As the Ca²⁺-release from non-ER compartments remained sensitive to ATP we speculate that mitochondria might be involved.